Continuous exposure to highly active antiretroviral therapy (HAART) is not without some treatment-related drawbacks: the high risk of drug-resistant viruses emerging in HIV-infected patients with poor adherence; the metabolic complications that can arise and that may become a leading cause of mortality in the near future; and, of course, the high economic cost of the treatment itself.1-3 Nowadays, prolonged treatment interruptions driven by CD4 count are being investigated in an attempt to mitigate these problems. In this situation, nadir CD4 count is the main predictor of CD4 count decrease.4,5 We have previously demonstrated that thymic volume is the best predictor of speed and degree of CD4 T-cell recovery in patients receiving HAART.6-8 Thus, in this new treatment interruption scenario, thymic volume might have an important role in CD4 T-cell loss.
Our main objective is to analyze the predictive capacity of thymic volume, along with other variables, in CD4 T-cell loss after prolonged treatment interruption in a group of immunologically preserved HIV-infected patients with nadir CD4 count greater than 250 cells/μL.
MATERIALS AND METHODS
In July 2003, the Viral Hepatitis and AIDS Study Unit began a project of CD4 count-guided HAART interruption in HIV-infected patients. The selection criteria for this cohort were as follows: (1) persistent undetectable plasma viral load (<50 copies/mL) for the preceding 12 months (or more); (2) CD4 count greater than 500 cells/μL at the moment of interruption; and (3) nadir CD4 count greater than 250 cells/μL. Up to the year 2004, 39 of 45 consecutive patients meeting these criteria agreed to participate and gave written informed consent. The study was approved by the hospital‘s ethical committee. Clinical, immunologic, and virologic examinations were performed at the moment of treatment interruption and every 4 weeks thereafter. Moreover, all patients included in this study underwent a thoracic computed tomography (CT) scan at baseline for thymic volume measurements.
For the purpose of this study, significant CD4 T-cell loss was considered as a decrease to less than 350 cells/μL in at least 2 consecutive time points during the follow-up, as recommended by current guidelines to resume treatment.9
CD4 count was determined in fresh samples by flow cytometry. Quantitative polymerase chain reaction (PCR) was used to measure plasma HIV-1 RNA (HIV-1 Monitor Test kit; Roche Molecular Systems, Hoffman-La Roche Ltd, Basel, Switzerland), according to the manufacturer's instructions. This assay has a detection limit of 50 HIV-1 RNA copies/mL. A modified PCR-based method adapted to a real-time PCR using a LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany) for quantifying the number of T-cell receptor excision circles (TRECs) per 10 million peripheral blood mononuclear cells (PBMCs) was used.10 Hepatitis C virus (HCV) RNA was measured by a commercially available PCR procedure (COBAS AMPLICOR; Roche Diagnostics, Barcelona, Spain). Determination of IL-7 levels in serum samples was performed using a high-sensitivity colorimetric enzyme-linked immunosorbent assay (QuantikineHS human IL-7 immunoassay kit; R and D Systems, Inc, Minneapolis, MN). This assay has a lower detection threshold of 0.1 pg/mL. Mediastinal CT was performed with a modified previously described method.10 Briefly, coded samples were always measured by the same radiologist, using a Philips Tomoscan AV PS in a blind analysis. With helical scanning, axial images were obtained using 5-mm-slice sections, pitch 1.4, starting at 30 seconds after the beginning of intravenous contrast administration from the external notch through the xiphoid. The area occupied by the thymus on each slice was delimited by selecting the contour of the tissue using 350 W, 50 L, and excluding mediastinal fat infiltration. The software EasyVision version 4.3 directly determined the thymic volume.
Continuous variables are expressed as median (interquartile range in parenthesis), and categorical ones are presented as number of cases (percentage in parenthesis). Kaplan-Meier curves were used for all time-to-event analyses. When 2 consecutive measurements of CD4 count were less than 350 cells/μL, only the earliest value was used for the statistical analysis. The comparison between groups in the univariate analysis was done using the log-rank test. Continuous variables included in this univariate study were categorized using the median as the cutoff value. All variables with significant association (P < 0.1) were analyzed as continuous variables by Cox proportional hazards regression model to compute the relative risk (95% confidence interval) and to determine variables independently associated with CD4 T-cell loss. Multivariate analysis was considered significant when P < 0.05. Mann-Whitney test was used to compare different variables between 2 groups of patients. Spearman rank correlation and Pearson product moment correlation were used to analyze the correlations between variables. This study was censored in May 2005. Statistical analysis was performed with the SPSS software (Statistical Package for the Social Sciences version 12.0; SPSS, Inc, Chicago, IL).
Baseline characteristics of the 39 patients who ceased antiretroviral treatment are summarized in Table 1. The median time-to-event was 48 (range, 28-56) weeks. Twenty-three percent (n = 9) of the patients reached the endpoint defined for this study (CD4 T-cell loss less than 350 cells/μL) during the follow-up, with the earliest event achieved after 8 weeks of treatment interruption. The median CD4 count decreased from 636 (range, 506-816) to 447 (range, 289-561) cells/μL from week 12 to week 80. Compared with baseline, the individuals in this study had lost, at week 12, a median of 277 (range, 137-617) cells/μL, and, by week 80, had lost 537 (345-1144) cells/μL. In addition, between weeks 16 and 80 post-treatment interruption, 5 patients reinitiated HAART (2 by thrombocytopenia and 3 by self-decision), and 5 other patients initiated HCV antiviral therapy (peginterferon plus ribavirin). All these patients were censored (omitted) beyond this point.
Univariate analysis showed that CD4 T-cell decline after treatment interruption was only significantly associated with thymic volume (P = 0.029), although it was almost significantly associated with nadir CD4 count (P = 0.057), as shown by Kaplan-Meier survival analysis curves in Figure 1. The probability of CD4 T-cell loss was not statistically associated (all with P > 0.1) with the following stratified variables: median CD4 count, TREC-bearing cell count, age, sex, HCV coinfection, or early viral load rebound (mean, 3.25 log10 copies/mL; range, 1.83-5.03 log10 copies/mL) at week 4.
During the first 48 weeks of treatment interruption, the group of patients with high thymic volume did not show a CD4 T-cell loss below 350 cells/μL. Only 10.5% (n = 2) of patients with high thymic volume versus 35.3% (n = 6) with low thymic volume had a decrease in their CD4 count below 350 cells/μL after 52 weeks of interruption. In this last group, the earliest event was achieved after 8 weeks of treatment interruption. No statistical differences were found between basal characteristics of the patients with low thymic volume (<5.6 cm3) and those of the patients with a high thymic volume (≥5.6 cm3), with the exception of age (P = 0.03), as shown in Table 1. Moreover, thymic volume was not associated with time to reach a viral load rebound greater than 10,000 copies/mL after treatment interruption.
Moreover, no correlation between thymic volume and CD4 count as continuous variables was found during the follow-up, with the exception of week 72 (P = 0.020). However, nadir CD4 count directly correlated with CD4 count with statistical significance consecutively from baseline to week 12, and then eventually during the rest of the follow-up (weeks 24 and 52).
Only thymic volume was independently associated with CD4 T-cell loss among the variables with P < 0.1 (thymic volume and nadir CD4 count) included as continuous variables in the multivariate Cox proportional hazards regression model (P = 0.04; relative risk, 0.76; 95% confidence interval, 0.59-0.99). Nadir CD4 count was not statistically associated (P = 0.15).
The results of this study show that HIV-infected patients with a CD4 T-cell nadir greater than 250 cells/μL and with high thymic volume (above median value) experienced a slower loss of CD4 T cells after halting treatment. To our knowledge, this is the first time that thymic volume has been observed to predict CD4 T-cell loss rates among persons halting HAART.
One limitation of this study is the sample size. However, it is important to highlight that our study was restricted, as previously recommended,4,5 to patients with CD4 count greater than or equal to 500 cells/μL and nadir CD4 count greater than 250 cells/μL.
We have previously demonstrated that thymic volume independently predicts CD4 T-cell recovery in patients treated with HAART.6-8 In the same way, we have shown that thymic volume is the best predictor of CD4 T-cell decline after treatment interruption (ie, patients with low thymic volume had faster CD4 T-cell loss). It is also remarkable that patients with high thymic volume did not undergo any event during the first 48 weeks after interruption, whereas 35.3% (n = 6) of the patients with low thymic volume had their CD4 T-cell level drop below 350 cells/μL during the same period of time. Nevertheless, neither age nor TREC-bearing cell count was associated with CD4 T-cell loss, probably because of the narrow age range of the patients (mean, 39 years; range, 37-43 years) and because TREC-bearing cell count might be influenced by either the immune activation or the peripheral dilution effect.
Despite a delayed CD4 count recovery among antiretroviral-naive HIV/HCV-coinfected patients receiving HAART that has been reported,11,12 we did not find any association between HCV coinfection and CD4 T-cell decline in this HAART interruption scenario. In treatment interruptions, it has been reported that nadir CD4 count is a good predictor of CD4 T-cell decline.4,5 Nevertheless, in this study, nadir CD4 count at the moment of HAART interruption was not associated with the event, probably because this study was performed in patients with a narrow nadir CD4 count ranging from 337 to 438 cells/μL. On the other hand, IL-7 levels are involved in T-cell homeostasis and regeneration. In the present study, CD4 count inversely correlated with IL-7 level only at baseline-losing it during the follow-up (data not shown).
In summary, data presented herewith show that the measurement of thymic volume might predict CD4 T-cell loss after treatment interruption in HIV-infected adults with high nadir CD4 count. Thus, this could be a useful tool to select patients who would be good candidates for prolonged HAART interruption.
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